Serial Observations on Patterns of Growth, Myelin Formation, Maintenance and Degeneration in Cultures of New-Born Rat and Kitten Cerebellum

New-born rat and kitten cerebellum may be maintained for prolonged periods (over 5 months) in the Maximow assembly if explanted on to a coverslip previously coated with a thin gel of reconstituted rat tail collagen and fed a glucose-enriched "natural" medium. After a 2 week period of adjustment and early outgrowth, most cultures exhibit myelin formation. Axons located within the surrounding neuroglial sheet of the explant area myelinate. The sheaths are first evident as long, unsegmented, smooth, parallel, refractile lines. Simultaneously, neuronal nuclei tend to assume central positions and powdery granules of Nissl substance and lipoid materials begin to accumulate within the cytoplasm. During prolonged maintenance, axons may increase in width and the myelin may thicken. Some exhibit transient irregularities and swellings. Degeneration of some axons occurs manifested either by (a) progressive swellings and distortions of the myelin sheath and thinning of intervening portions of the axons which finally yield, leaving the swellings as myelin bodies, or by (b) small aneurysm-like distortions of myelin sheaths on thinning axons which become dull, irregular, and thread-like filaments beaded by the former herniations. The observations are compared with previous studies of in vitro and in vivo myelin formation with particular reference to neuronal-neuroglial relationships.     

Myelin sheath survival after guanethidine-induced axonal degeneration

Membrane-membrane interactions between axons and Schwann cells are required for initial myelin formation in the peripheral nervous system. However, recent studies of double myelination in sympathetic nerve have indicated that myelin sheaths continue to exist after complete loss of axonal contact (Kidd, G. J., and J. W. Heath. 1988. J. Neurocytol. 17:245-261). This suggests that myelin maintenance may be regulated either by diffusible axonal factors or by nonaxonal mechanisms. To test these hypotheses, axons involved in double myelination in the rat superior cervical ganglion were destroyed by chronic guanethidine treatment. Guanethidine-induced sympathectomy resulted in a Wallerian-like pattern of myelin degeneration within 10 d. In doubly myelinated configurations the axon, inner myelin sheath (which lies in contact with the axon), and approximately 75% of outer myelin sheaths broke down by this time. Degenerating outer sheaths were not found at later periods. It is probably that outer sheaths that degenerated were only partially displaced from the axon at the commencement of guanethidine treatment. In contrast, analysis of serial sections showed that completely displaced outer internodes remained ultrastructurally intact. These internodes survived degeneration of the axon and inner sheath, and during the later time points (2-6 wk) they enclosed only connective tissue elements and reorganized Schwann cells/processes. Axonal regeneration was not observed within surviving outer internodes. We therefore conclude that myelin maintenance in the superior cervical ganglion is not dependent on direct axonal contact or diffusible axonal factors. In addition, physical association of Schwann cells with the degenerating axon may be an important factor in precipitating myelin breakdown during Wallerian degeneration.     

Dysmyelination, demyelination and reactive astrogliosis in the optic nerve of the taiep rat

Taiep is an autosomal recessive mutant rat that shows a highly hypomyelinated central nervous system (CNS). Oligodendrocytes accumulate microtubules (MTs) in association with endoplasmic reticulum (ER) membranes forming MT-ER complexes. The microtubular defect in oligodendrocytes, the abnormal formation of CNS myelin and the astrocytic reaction were characterized by immunocytochemical and ultrastructural methods during the first year of life. Optic nerves of both control and taiep rats were processed by the immunoperoxidase method using antibodies against tubulin, myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). Taiep oligodendrocytes are strongly immunoreactive against tubulin, indicative of a significant accumulation of microtubules. Early differentiated oligodendrocytes observed with electron microscopy show that MT-ER complexes are mainly present in the cell body. This defect increases during the first year of life; oligodendrocytes show large MT-ER complexes projected within oligodendrocyte processes. Using anti-MBP, there was a progressive reduction of immunolabeling in the myelin sheaths as taiep rats grew older. Ultrastructural analysis revealed severely dysmyelinated axons with a frequently collapsed periaxonal collar. However, through age the myelin sheath became gradually infiltrated by MTs, suggesting their contribution to premature loss of myelin in the taiep rat. Axons of one-year-old taiep rats were severely demyelinated. Modifications in astrocytes revealed by the GFAP antibody showed a strong hypertrophy with increased immunostaining in their processes. As demyelination of axons progressed, taiep rats developed a strong astrogliosis. The present findings suggest that in taiep rats the early abnormal myelination of axons affects the adequate maintenance of myelin, leading to a progressive loss of myelin components and severe astrogliosis, features that should be considered in the pathogenesis of dysmyelinating diseases.     

Axonal Transport of Choline Lipids in Normal and Regenerating Rat Sciatic Nerve

Abstract: Biochemical methods were used to study the time course of transport of choline phospholipids (labeled by the injection of [³H]choline into the ventral horn of the lumbar spinal cord) in rat sciatic nerve. Autoradiographic methods were used to localize the transported lipid within motor axons. Transported phospholipid, primarily phosphatidylcholine, present in the nerve at 6 h, continued to accumulate over the following 12 days. No discrete waves of transported lipid were observed (a small wave of radioactive phospholipid moving at the high rate would have been missed); the amounts of radioactive lipid increased uniformly along the entire sciatic nerve. In light-microscope autoradiographs, a class of large-caliber axons, presumably motor axons, retained the labeled lipid. Some lipid, even at 6 h, was seen within the myelin sheaths. Later, the labeling of the myelin relative to axon increased. The continued accumulation of choline phospholipids in the axons probably signifies their prolonged release from cell bodies and their retention in various axonal membranes, including the axolemma. The build-up of these phospholipids in myelin probably represents their transfer from the axons to the myelin sheaths surrounding them. When nerves are crushed and allowed to regenerate for 6 or 12 days, choline phospholipids transported during these times enter the regenerating nerve. In light and electron microscope autoradiographs, transported lipid was seen to be localized primarily in the regenerating axons. However, grains overlay the adjacent Schwann cell cytoplasm, indicating transported lipids were transferred from the regenerating axons to the associated Schwann cells. In addition, some cells not associated with growing axons were labeled, suggesting that phosphatidylcholine and possibly acetylcholine, carried to the regenerating axons by axonal transport, were actively metabolized in the terminal, with released choline label being used by other cells. These results demonstrate that axonal transport supplies mature and growing axons and their glial cells with choline phospholipids.     

THE FINE STRUCTURE OF ACOUSTIC GANGLIA IN THE RAT

Nerve cell bodies in the spiral and vestibular ganglia of the adult rat are surrounded by thin (about ten lamellae) myelin sheaths which differ in several respects from typical axonal myelin. In some instances lamellae surrounding perikarya appear as typical major dense lines, and in others as thin Schwann cell sheets in which cytoplasm persists. Discontinuities and irregularities appear in the structure of perikaryal myelin. Lamellae may terminate anywhere within the sheaths; they may bifurcate; they may reverse their direction; or they may merge with each other. The number of lamellae varies from one part of a sheath to another. In addition, the myelin of a single perikaryal sheath may receive contributions from more than one Schwann cell, which overlap and interleave with each other. The ganglion cells are of two types: those which are densely packed with the usual cytoplasmic organelles but have few neurofilaments (granular neurons), and those which exhibit large areas containing few organelles but have a high concentration of neurofilaments (filamented neurons). The latter cell type is ensheathed by myelin which is generally more compact that that surrounding the former. The formation and the physiologic significance of perikaryal myelin are discussed.     

Progressive white matter pathology in the spinal cord of transgenic mice expressing mutant (P301L) human tau

Transgenic mice expressing mutant (P301L) tau develop paresis, neurofibrillary tangles and neuronal loss in spinal motor neurons beginning at 4 to 6 months of age. Astrocytes and oligodendrocytes acquire filamentous tau inclusions at later ages. Here we report pathology in the spinal white matter of these animals. Progressive white matter pathology, detected as early as 2 months of age, was most marked in lateral and anterior columns, with sparing of posterior columns until late in the disease. Early changes in Luxol fast blue/periodic acid Schiff (LFB/PAS) and toluidine blue stained sections were vacuolation of myelin followed by accumulation of myelin figures within previous axonal tubes and finally influx of PAS-positive macrophages. Myelin debris and vacuoles were found in macrophages. At the ultrastructural level, myelinated axons showed extensive vacuolation of myelin sheaths formed by splitting of myelin lamellae at the intra-period line, while axons were atrophic and contained densely packed neurofilaments. Other axons were lost completely, resulting in collapse and phagocytosis of myelin sheaths. Also present were spheroids derived from swollen axons with thin myelin sheaths containing neurofilaments, tau filaments and degenerating organelles. Many oligodendrocytes had membrane-bound cytoplasmic bodies composed of tightly stacked lamellae capped by dense material. The vacuolar myelopathy in this model to some extent resembles that reported in acquired immune deficiency syndrome and vitamin B12 deficiency. The progressive axonal pathology is most consistent with a dying-back process caused by abnormal accumulation of tau in upstream neurons, while vacuolar myelinopathy may be a secondary manifestation of neuroinflammation.     

REDUNDANT MYELIN SHEATHS AND OTHER ULTRASTRUCTURAL FEATURES OF THE TOAD CEREBELLUM

Some of the myelin sheaths in the cerebellum of normal adult toads exhibit extensive evaginations of their full thickness. These redundant flaps of myelin are collapsed; i.e., they contain no axon and have no lumen. They extend away from the parent axonal myelin sheaths and tend to enfold other myelinated fibers or granule cell perikarya, producing bizarre configurations of myelin and what appear to be partially or completely myelinated cell bodies. In some instances, only the redundant flap of myelin appears in the plane of section, and its attachment to an axonal myelin sheath in another plane is only inferred. Single lamellae of myelin also tend to invest cerebellar granule cells and other processes, and these too appear to fold on themselves producing two- or four-layered segments. It is suggested that there are two phases of myelinogenesis: an initial "wrapping" phase, followed by a prolonged second phase during which internodes of myelin increase in both length and girth by a process other than wrapping, and that the occurrence of redundant myelin sheaths may reflect overgrowth of myelin during the second phase. Observations on the general organization of the toad cerebellum and on the ultrastructural cytology of its layers are also presented.     

Progesterone as a neurosteroid: Actions within the nervous system

Summary 1. Some progesterone is synthesized within both the central and the peripheral nervous systems, where it regulates neurotransmission and important glial functions, such as the formation of myelin. Progesterone can thus be designated a neurosteroid.2. Steroids act not only on the brain, but also on peripheral nerves, which offer many advantages to study the biological significance of locally produced neurosteroids: their remarkable plasticity and regenerative capacity and their relatively simple structure.3. By using the regenerating mouse sciatic nerve as a model, we have shown that progesterone synthesized by rat Schwann cells promotes the formation of new myelin sheaths. Progesterone also increases the number of myelinated axons when added at a low concentration to cocultures of Schwann cells and sensory neurons.4. These findings show a function on myelination for locally produced progesterone and suggest a new pharmacological approach of myelin repair.     

Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination

Some steroids are synthesized within the central and peripheral nervous system, mostly by glial cells. These are known as neurosteroids. In the brain, certain neurosteroids have been shown to act directly on membrane receptors for neurotransmitters. For example, progesterone inhibits the neuronal nicotinic acetylcholine receptor, whereas its 3alpha,5alpha-reduced metabolite 3alpha, 5alpha-tetrahydroprogesterone (allopregnanolone) activates the type A gamma-aminobutyric acid receptor complex. Besides these effects, neurosteroids also regulate important glial functions, such as the synthesis of myelin proteins. Thus, in cultures of glial cells prepared from neonatal rat brain, progesterone increases the number of oligodendrocytes expressing the myelin basic protein (MBP) and the 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). An important role for neurosteroids in myelin repair has been demonstrated in the rodent sciatic nerve, where progesterone and its direct precursor pregnenolone are synthesized by Schwann cells. After cryolesion of the male mouse sciatic nerve, blocking the local synthesis or action of progesterone impairs remyelination of the regenerating axons, whereas administration of progesterone to the lesion site promotes the formation of new myelin sheaths.     

Myelin abnormalities in mice deficient in galactocerebroside and sulfatide

Myelin sheath formation depends on appropriate axo-glial interactions that are mediated by myelin-specific surface molecules. In this study, we have used quantitative morphological analysis to determine the roles of the prominent myelin lipids galactocerebroside (GalC) and sulfatide in both central and peripheral myelin formation, exploiting mutant mice incapable of synthesizing these lipids. Our results demonstrate a significant increase in uncompacted myelin sheaths, the frequency of multiple cytoplasmic loops, redundant myelin profiles, and Schmidt-Lanterman incisures in the CNS of these mutant mice. In contrast, PNS myelin appeared structurally normal in these animals; however, at post-natal day 10, greater than 10% of the axons withered and pulled away from their myelin sheaths. These results indicate that GalC and sulfatide are critical to the formation of CNS myelin. In contrast, PNS myelin formation is not dependent on these lipids; however, GalC and sulfatide appear to be instrumental in maintaining Schwann cell-axon contact during a specific developmental window.     

N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination

Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation.     

THE GEOMETRY OF PERIPHERAL MYELIN SHEATHS DURING THEIR FORMATION AND GROWTH IN RAT SCIATIC NERVES

In rat sciatic nerves, a small bundle of fibers was identified in which myelin sheaths were absent at birth, appeared within 3 days, and grew rapidly for 2 wk. During this interval, nerves were removed from littermates and were sectioned serially in the transverse plane. Alternating sets of thin and thick sections were used to prepare electron micrograph montages in which single myelinating axons could be identified and traced distally. During the formation of the first spiral turn, the mesaxon's length and configuration varied when it was studied at different levels in the same Schwann cell. The position of the mesaxon's termination shifted while its origin, at the Schwann cell surface, remained relatively constant. Along myelin internodes composed of two to six spiral turns, there were many variations in the number of lamellae and their contour. Near the mesaxon's origin, longitudinal strips of cytoplasm separated the myelin layers. Thicker sheaths were larger in circumference, more circular in transverse sections, and more uniform at different levels. Irregularities were confined to the paranodal region, and separation of lamellae by cytoplasm occurred at Schmidt-Lantermann clefts. Approximate dimensions of the bundle, its largest fibers, and their myelin sheaths were measured and calculated. The myelin membrane's transverse length and area increased exponentially with time; the growth rate increased rapidly during the formation of the first four to six spiral layers and remained relatively constant during the subsequent enlargement of the compact sheath.     

The demonstration by transplantation of the very restricted remyelinating potential of post-mitotic oligodendrocytes

To examine the remyelinating ability of post-mitotic oligodendrocytes, we subjected cell preparations derived from neonatal and adult rats to 40 Grays of X-irradiation to remove mitotically active cells and injected them into areas of demyelination in which the inherent ability to generate remyelinating cells had been inhibited. The extensive remyelination seen following implantation of non-irradiated neonatal and adult cells was almost completely abolished when the transplanted cell suspension was exposed to 40 Grays of X-irradiation, demonstrating that effective remyelination requires the generation of cells by mitosis. Radiation-resistant and therefore non-dividing oligodendrocytes were detected in areas of demyelination following transplantation of neonatal cultures and oligodendrocyte preparations derived from the adult nervous system. However, the pattern of myelin formation associated with the radiation-resistant oligodendrocytes from the two sources was different. Following implantation of X-irradiated neonatal cultures, a small number of oligodendrocytes could be found within the area of demyelination, and although these cells formed sheets of myelin membrane, they did not form myelin sheaths. After implantation of X-irradiated adult cells, in addition to the aberrant myelin formation seen with the neonatal cells, some myelin sheaths were observed. Our findings confirm that effective remyelination requires cell division and suggest that there may be diverse populations of radiation-resistant oligodendrocytes in the adult nervous system, some of which can form myelin sheaths and others of which can only make myelin sheets. Important for the interpretation of our previous studies is the demonstration here that 40 Grays of X-irradiation per se does not inhibit oligodendrocytes from remyelinating axons.     

Myelin formation in cultures of previously dissociated mouse spinal cord

Myelin formation in cultures of previously dissociated spinal cord from foetal mice is described. In addition to the expected pattern of myelination, in which axons are closely wrapped by myelin lamellae, redundant folds of myelin have been found, as have double sheaths surrounding a single axon. Hypotheses concerning the generation of these appearances are discussed. It is suggested that certain intracytoplasmic laminar bodies found in oligodendrocytes in vitro may be of mitochondrial origin.     

Immunocytochemical localization of carbonic anhydrase in myelinated fibers in peripheral nerves of rat and mouse

Rat sciatic nerve, spinal root, and cranial nerve were immunostained with an antibody against rat brain carbonic anhydrase II (ca), to determine the localization of ca in the rat peripheral nervous system (PNS). Similar methods were applied to mouse nerves to see if that antigen could be detected in the PNS of this species. In rat nerves, intense immunostaining was observed in the axoplasm of many of the myelinated fibers, whereas others were stained less intensely or were negative. A heterogeneous pattern of immunostaining was also found in neuronal perikarya within the ganglia, and in some regions of the ganglia ca immunostaining was found in putative satellite cells and their processes. Ca in rat PNS therefore appears to occur at both neuronal and glial sites, whereas it is exclusively glial in the CNS. In longitudinal sections of some fibers within rat nerves, ca immunostaining could be detected at the inner boundaries of the myelin sheaths. In mouse nerves, axoplasmic staining was observed but it was fainter than in rat nerves. Interspecies differences were most obvious in the dorsal columns of the spinal cord. In rat, intensely stained axons proceeded through the roots into the gracilis or cuneate and often into the gray matter. In mouse, there was much less immunostaining of axons but more intense ca immunostaining in CNS myelin than in the CNS myelin in the rat cord. The implications concerning putative functions of ca in the rodent nervous system are discussed.     

Myelin-associated glycoprotein and myelinating Schwann cell-axon interaction in chronic B,B''-iminodipropionitrile neuropathy

The myelin-associated glycoprotein (MAG) is a heavily glycosylated integral membrane glycoprotein which is a minor component of isolated rat peripheral nervous system (PNS) myelin. Immunocytochemically MAG has been localized in the periaxonal region of PNS myelin sheaths. The periaxonal localization and biochemical features of MAG are consistent with the hypothesis that MAG plays a role in maintaining the periaxonal space of myelinated fibers. To test this hypothesis, MAG was localized immunocytochemically in 1-micron sections of the L5 ventral root from rats exposed to B,B'-iminodipropionitrile. In chronic states of B,B'-iminodipropionitrile intoxication, Schwann cell periaxonal membranes and the axolemma invaginate into giant axonal swellings and separate a central zone of normally oriented axoplasm from an outer zone of maloriented neurofilaments. Ultrastructurally, the width of the periaxonal space (12-14 nm) in the ingrowths is identical to that found in normally myelinated fibers. These Schwann cell ingrowths which are separated from compact myelin by several micra are stained intensely by MAG antiserum. Antiserum directed against Po protein, the major structural protein of compact PNS myelin, does not stain the ingrowths unless compact myelin is present. These results demonstrate the periaxonal localization of MAG and support a functional role for MAG in maintaining the periaxonal space of PNS myelinated fibers.     

Inhibition of myelin membrane sheath formation by oligodendrocyte-derived exosome-like vesicles

Myelin formation is a multistep process that is controlled by a number of different extracellular factors. During the development of the central nervous system (CNS), oligodendrocyte progenitor cells differentiate into mature oligodendrocytes that start to enwrap axons with myelin membrane sheaths after receiving the appropriate signal(s) from the axon or its microenvironment. The signals required to initiate this process are unknown. Here, we show that oligodendrocytes secrete small membrane vesicles, exosome-like vesicles, into the extracellular space that inhibit both the morphological differentiation of oligodendrocytes and myelin formation. The inhibitory effects of exosome-like vesicles were prevented by treatment with inhibitors of actomyosin contractility. Importantly, secretion of exosome-like vesicles from oligodendrocytes was dramatically reduced when cells were incubated by conditioned neuronal medium. In conclusion, our results provide new evidence for small and diffusible oligodendroglial-derived vesicular carriers within the extracellular space that have inhibitory properties on cellular growth. We propose that neurons control the secretion of autoinhibitory oligodendroglial-derived exosomes to coordinate myelin membrane biogenesis.     

Fine structure of the retino-optic nerve junction in the chicken

The aim of this study is to investigate a fine structure of the retino-optic nerve junction in the chicken. We especially focused on the myelin sheaths and astrocytes in the intraocular optic nerve (ION) and its adjoining parts. A part of the axons of retinal nerve fiber layer (NFL) were myelinated. Ganglion cell axons were ensheathed by loose myelin in the NFL and by a compact one in the ION and optic nerve (ON). Myelin structure changed from loose type to a compact one within the very narrow NFL-ION junction. Loose myelin forming cells are dark type of oligodendrocytes in the retina. From the most peripheral ON to the choroidal part of ION, astrocytes contained abundant microtubules. The optic nerve around the lamina cribrosa is exposed to mechanical force during eye movement. It is suggested that these microtubules may perform the cytoskeletal function. Astrocytes in the retinal part of ION had longer processes filled with abundant gliofilaments. They may provide the mechanical support for the ganglion cell axons, which are exposed directly to intraocular pressure. Although astrocytes in the retinal level of ION extended their processes into the retina, their soma was never found in the retina.     

Adult stem cells and multiple sclerosis

Multiple sclerosis (MS) is a common neurological disease and a major cause of disability, particularly affecting young adults. It is characterized by patches of damage occurring throughout the brain and spinal cord, with loss of myelin sheaths - the insulating material around nerve fibres that allows normal conduction of nerve impulses - accompanied by loss of cells that make myelin (oligodendrocytes). In addition, we now know that there is damage to nerve cells (neurones) and their fibres (axons) too, and that this occurs both within these discrete patches and in tissue between them. The cause of MS remains unknown, but an autoimmune reaction against oligodendrocytes and myelin is generally assumed to play a major role, and early acute MS lesions almost invariably show prominent inflammation. Efforts to develop cell therapy in MS have long been directed towards directly implanting cells capable of replacing lost oligodendrocytes and regenerating myelin sheaths. Accordingly, the advent of techniques to generate large numbers of oligodendrocytes from embryonic stem cells appeared a significant step towards new stem cell treatments for MS; while the emerging consensus that adult stem cells from, for example, the bone marrow had far less potential to turn into oligodendrocytes was thought to cast doubt on their potential value in this disease. A number of scientific and medical concerns, not least the risk of tumour formation associated with embryonic stem cells, have however, prevented any possible clinical testing of these cells in patients. More recently, increasing understanding of the complexity of tissue damage in MS has emphasized that successful cell therapy may need to achieve far more than simply offering a source of replacement myelin-forming cells. The many and varied reparative properties of bone marrow-derived (mesenchymal) stem cells may well offer new and attractive possibilities for developing cell-based treatments for this difficult and disabling condition.